Homologous Recombination & Human Cell Radiosensitivity

描述(申请人提供)：染色体稳定性和对电离辐射(IR)的抗性要求同源重组国家修复(HRR)的完整性，该修复作用于已复制的染色体区域产生的坦率双链断裂(DSB)。HRR对于恢复当DMA复制分叉崩溃时产生的单侧DSB也是至关重要的，甚至可以通过促进阻断氧化损伤的非突变旁路来拯救阻塞的分叉。在非人类脊椎动物细胞中，RAD51同源基因(XRCC2/3和RAD51B/C/D)的突变赋予了相似的表型，具有中等的IR敏感性和高度的染色体不稳定性。因此，这五种蛋白质为研究HRR的分子性质提供了一个框架。该项目使用CHO仓鼠和人类细胞系的敲除突变体来确定HRR提高染色体稳定性和抗辐射能力的机制。通过RAD51D基因敲除CHO细胞的特征，特定目的1测试了HRR抑制癌症相关类型的基因改变的假设。HPRT基因座的基因突变率以及DHFR和CAD基因座的基因扩增将被量化，并将表征HPRT突变谱。特异性目标2将在TP53-正常的永生化二倍体人成纤维细胞中构建XRCC3的零突变系，并评估它们在辐射敏感性、染色体畸变和其他终点方面的基因组不稳定性。具体目标3将确定HRR在细胞周期中对仓鼠和人类细胞IR抵抗变化的贡献。要检验的假设是：(A)经典的S期抗性是由于HRR；(B)当未修复的损伤稍后由DMA复制机制处理时，HRR有助于G1期照射的细胞存活；(C)IR诱导的Hprt突变在S期的产率低于G1期，这是因为HRR在S期促进双链断裂的无错误修复。这些综合研究可能会为癌症放射治疗提供一个更合理的基础，并为HRR如何防止内源性过程和外源性因素启动癌变提供深入的见解。

DESCRIPTION (provided by applicant): Chromosome stability and resistance to ionizing radiation (IR) require the integrity of homologous recombine- national repair (HRR), which acts on frank double-strand breaks (DSBs) produced in the already replicated chromosomal regions. HRR is also crucial for restoring one-sided DSBs arising when DMA replication forks collapse, and may even act by rescuing blocked forks by facilitating non-mutagenic bypass of blocking oxidative lesions. In non-human vertebrate cells, mutations in the Rad51 paralogs (XRCC2/3 & Rad51B/C/D) confer similar phenotypes of moderate IR sensitivity and high chromosome instability. Thus, these five proteins provide a framework for studying the molecular nature of HRR. This project uses knockout mutants of CHO hamster and human cell lines to identify mechanisms by which HRR promotes chromosome stability and radiation resistance. Specific Aim 1 tests the hypothesis that HRR suppresses cancer-associated types of genetic alterations, as revealed by characterizing Rad51d knockout CHO cells. Rates of gene mutation at the hprt locus and gene amplification at the dhfr and CAD loci will be quantified, and the hprt mutation spectrum will be characterized. Specific Aim 2 will construct null mutant lines of XRCC3 in Tp53-normal immortalized diploid human fibroblasts and assess their genomic instability with respect to radiosensitivity, chromosomal aberrations, and other endpoints. Specific Aim 3 will determine the contribution of HRR to changes in IR resistance during the cell cycle in hamster and human cells. The hypotheses to be tested in this aim are: (a) Classical S phase resistance is due to HRR; (b) HRR contributes to the survival of cells irradiated in G1 phase when unrepaired damage is later processed by the DMA replication machinery; (c) The yield of IR-induced hprt mutations is lower in S phase than in G1 phase because HRR acts during S phase to promote error-free repair of DSBs. These integrated studies may lead to a more rational basis for cancer radiotherapy and bring insights into how HRR prevents the initiation of carcihogenesis by endogenous processes and exogenous agents.